An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

Abstract

Source: Riccione, K., et al. Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care. J. Vis. Exp. (2015).

This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host genome and produce surface-bound CARs targeting the tumor antigen.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Splenectomy and T Cell Preparation

Day 0:

1. Pour 10 ml of T cell media (TCM) into a 50 ml conical and place on ice for spleen collection.
2. Sacrifice the appropriate number of animals by CO₂ asphyxiation and secondary decapitation: Place animals in a cage receiving CO₂ at a flow rate of 10-30% cage volume/minute, per American Veterinary Medical Association guidelines (no more than 5 animals can be sacrificed sim....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Concanavalin A	Sigma Aldrich	C2010	Non-specific mitogen to induce T cell proliferation and viral transduction
RPMI 1640	Life Technologies	11875-093	T cell culture media
Bovine Serum Albumin (BSA), Fraction V—Standard Grade	Gemini Bio Products	700-100P	Blocks non-specific binding of retrovirus to retronectin-coated plates
Pharm Lyse (10X concentrate)	BD Biosciences	555899	Lyses red blood cells during splenocyte processing
70 µm Sterile Cell Strainers	Corning	352350	Filters away large tissue particles during splenocyte processing
100 mm BioCoat Culture Dishes with Poly-D-Lysine	Corning	356469	Promotes HEK293 cell adhesion to maximize proliferation after transfection
Dimethyl Sulfoxide	Sigma Life Sciences	D2650	Necessary for complete dissolution of temozolomide
Saline	Hospira	IM 0132 (5/04)	Solvent for temozolomide and ketamine/xylazine
Ketathesia HCl	Henry Schein Animal Health	11695-0701-1	Ketamine solution
AnaSed	Lloyd Inc	N/A	Xylazine sterile solution 100 mg/mL